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. Author manuscript; available in PMC: 2019 Sep 9.
Published in final edited form as: J Mol Cell Cardiol. 2018 Jul 5;121:107–123. doi: 10.1016/j.yjmcc.2018.07.003

Fig. 3.

Fig. 3.

TWEAK induces its own expression and that of its receptor (TWEAKR) via TRAF3IP2 and its downstream signaling intermediates NF-κB and AP-1 in cardiac fibroblasts. TWEAK (10 ng/ml) induced TWEAK expression in a time-dependent manner in CF, and lentiviral TRAF3IP2 knockdown (moi0.5 for 48 h) inhibited this effect (A; time ‘0’ represents no exogenous addition of TWEAK). Further, TWEAK upregulated TWEAKR expression in a time-dependent manner, an effect also blunted by TRAF3IP2 knockdown (B). Immunofluorescence revealed low levels of TWEAKR expression at basal conditions, and its increased levels after TWEAK treatment (10 ng/ml for 2 h), localized predominantly in the membrane at cytoplasm, and its reduced levels following TRAF3IP2 knockdown (C; arrows denote membrane and cytoplasmic localization of TWEAKR). Moreover, pretreatment with the p38 MAPK-specific inhibitor SB239063 (10 μM in DMSO for 1 h) and lentiviral-mediated p65 (moi0.5 for 48 h) and c-Jun (moi0.5 for 48 h) knockdown each attenuated TWEAK autoregulation (2 h) and its receptor upregulation (2 h; D). Knockdown of p65 and c-Jun was confirmed by immunoblotting and is shown on the right in panel E. gp130 served as an off-target. Further, silencing TRAF3IP2, p65 and c-Jun (moi0.5 for 48 h) attenuated TWEAK-induced TWEAK (F) and TWEAKR (G) promoter-reporter activities (both TWEAK and TWEAKR reporters contain 3 kb DNA fragments upstream of transcription start site and 5’-UTR). Importantly, adenoviral transduction of TRAF3IP2, but not control GFP, increased TRAF3IP2 protein expression in a dose-dependent manner (moi1–100 for 24 h) (H), and at a moi50, induced TWEAK and its receptor expression (I). A, B, D, F, H, I: A representative image from three independent immunoblotting experiments with similar results is shown on the left and summarized semi-quantification of intensity of immunoreactive bands by densitometry from all three experiments on the right. F, G, n = 6. C, Magnification: 40×. *P < at least 0.05 versus untreated, †P < 0.05 versus TWEAK, §P < 0.05 (n = 3–6).